Photoselection of nucleic acid ligands

Abstract

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to bFGF. Included in the invention are specific DNA ligands to bFGF identified by the photoSELEX method. Also included is a method for determining the position of a nucleic acid ligand-protein photoadduct.

What is claimed is:  
     
       1. A method for detecting the presence or absence of a peptide or protein target molecule in a sample which may contain said target molecule comprising: 
       a) exposing said sample which may contain said target molecule to a non-naturally occurring photoaptamer of said target molecule, under conditions wherein a target molecule:photoaptamer complex is formed if said target molecule is present, wherein said complex is not due to Watson/Crick base pairing;  
       b) irradiating said complexes, wherein said target molecule and photoaptamer photocrosslink; and  
       c) determining whether said target molecule:photoaptamer crosslink is formed, thereby detecting the presence or absence of the target molecule in the sample.  
     
     
       2. The method of  claim 1  wherein said photoaptamer is a single-stranded nucleic acid ligand. 
     
     
       3. The method of  claim 2  wherein said single-stranded nucleic acid ligand is ribonucleic acid. 
     
     
       4. The method of  claim 2  wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid. 
     
     
       5. The method of  claim 1  wherein said photoaptamer is labeled. 
     
     
       6. The method of  claim 5  wherein said label is a radiolabel. 
     
     
       7. The method of  claim 1  wherein said protein is not known to bind nucleic acids as part of its biological function. 
     
     
       8. The method of  claim 1  wherein said target molecule is a controlled substance. 
     
     
       9. The method of  claim 1  wherein said target molecule is a metabolite. 
     
     
       10. The method of  claim 1  wherein said sample is a biological substance. 
     
     
       11. The method of  claim 1  wherein said detection comprises 
       a) separating the target and the photoaptamer to a level sufficient for amplification of the photoaptamer by PCR;  
       b) amplifying the photoaptamer by PCR; and  
       c) detecting the amplified photoaptamer; whereby the presence or absence of the target molecule in the sample is detected.  
     
     
       12. A method for detecting the presence or absence of a protein or peptide target molecule in a sample which may contain said target molecule comprising: 
       a) identifying a photoaptamer from a candidate mixture of photoaptamers, said photoaptamer being a ligand of said target molecule, by a method comprising:  
       i) contacting the candidate mixture with said target molecule, wherein photoaptamers having an increased affinity to said target molecule relative to the candidate mixture form photoaptamer-target complexes;  
       ii) irradiating said complexes, wherein said target molecule and photoaptamer photocrosslink;  
       iii) partitioning the photocrosslinked photoaptamer-target complexes from the remainder of the candidate mixture; and  
       iv) amplifying the photocrosslinked photoaptamers to yield a ligand-enriched mixture of photoaptamers, whereby an increased affinity photoaptamer may be identified;  
       b) exposing said sample which may contain said target molecule to said increased affinity photoaptamer of said target molecule under conditions wherein a target molecule:photoaptamer complex is formed if said target molecule is present;  
       c) irradiating said complex, wherein said target molecule and photoaptamer photocrosslink; and  
       d) determining whether said target molecule:photoaptamer photocrosslink is formed, thereby detecting the presence or absence of the target molecule in the sample.  
     
     
       13. The method of  claim 12  further comprising: 
       iv) repeating steps i), ii), iii) and iv).  
     
     
       14. The method of  claim 12  wherein said photoaptamer is a single-stranded nucleic acid ligand. 
     
     
       15. The method of  claim 14  wherein said single-stranded nucleic acid ligand is ribonucleic acid. 
     
     
       16. The method of  claim 14  wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid. 
     
     
       17. The method of  claim 12  wherein said protein is not known to bind nucleic acids as part of its biological function. 
     
     
       18. The method of  claim 12  wherein said target molecule is a controlled substance. 
     
     
       19. The method of  claim 12  wherein said target molecule is a metabolite. 
     
     
       20. The method of  claim 12  wherein said sample is a biological substance. 
     
     
       21. The method of  claim 12  wherein said detection comprises 
       a) separating the target and the photoaptamer to a level sufficient for amplification of the photoaptamer by PCR;  
       b) amplifying the photoaptamer by PCR; and  
       c) detecting the amplified photoaptamer; whereby the presence or absence of the target molecule in the sample is detected.  
     
     
       22. The method of  claim 12  wherein said candidate mixture is prepared by synthesis from a template comprising a region of conserved sequence and a region of randomized and/or biased sequence. 
     
     
       23. The method of  claim 12  wherein said candidate mixture comprises nucleic acids each comprising a region of conserved sequence and a region of randomized sequence. 
     
     
       24. A method for measuring the amount of a peptide or protein target molecule in a sample which may contain said target molecule comprising: 
       a) exposing said sample which may contain said target molecule to a photoaptamer of said target molecule, under conditions wherein a target molecule:photoaptamer complex is formed;  
       b) irradiating said complex, wherein said target molecule and photoaptamer photocrosslink;  
       c) determining whether said target molecule:photocrosslink is formed; and  
       d) measuring the amount of the photoaptamer in the sample; whereby the amount of target molecule in the sample may be measured.  
     
     
       25. The method of  claim 24  wherein said photoaptamer is a single-stranded nucleic acid ligand. 
     
     
       26. The method of  claim 25  wherein said single-stranded nucleic acid ligand is ribonucleic acid. 
     
     
       27. The method of  claim 25  wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid. 
     
     
       28. The method of  claim 24  wherein said photoaptamernucleic acid ligand is labeled. 
     
     
       29. The method of  claim 28  wherein said label is a radiolabel. 
     
     
       30. The method of  claim 24  wherein said protein is not known to bind nucleic acids as part of its biological function. 
     
     
       31. The method of  claim 24  wherein said target molecule is a controlled substance. 
     
     
       32. The method of  claim 24  wherein said target molecule is a metabolite. 
     
     
       33. The method of  claim 24  wherein said sample is a biological substance. 
     
     
       34. The method of  claim 24  wherein said detection is achieved by PCR amplification of said nucleic acid ligand. 
     
     
       35. The method of  claim 24  wherein said measurement is qualitative. 
     
     
       36. The method of  claim 24  wherein said measurement is quantitative. 
     
     
       37. A method for measuring the amount of a peptide or protein target molecule in a sample which may contain said target molecule comprising: 
       a) identifying a photoaptamer from a candidate mixture of photoaptamers, said photoaptamer being a ligand of said target molecule, by a method comprising:  
       i) contacting the candidate mixture with said target molecule, wherein photoaptamers having an increased affinity to said target molecule relative to the candidate mixture form photoaptamer-target complexes;  
       ii) irradiating said complexes, wherein said target molecule and photoaptamer photocrosslink;  
       iii) partitioning the crosslinked target molecule and photoaptamer from the remainder of the candidate mixture; and  
       iv) amplifying the crosslinked photoaptamer to yield a ligand-enriched mixture of photoaptamers, whereby an increased affinity photoaptamer may be identified;  
       b) exposing said sample which may contain said target molecule to said increased affinity photoaptamer of said target molecule under conditions wherein a target molecule:photoaptamer complex is formed;  
       c) irradiating said complex, wherein said target molecule and photoaptamer photocrosslink;  
       d) determining whether said target molecule:photoaptamer crosslinked complex is formed; and  
       e) measuring the amount of the photoaptamer in the sample; whereby the amount of target molecule in the sample may be measured.  
     
     
       38. The method of  claim 37  further comprising: 
       iv) repeating steps i), ii), iii) and iv).  
     
     
       39. The method of  claim 37  wherein said photoaptamer is a single-stranded nucleic acid ligand. 
     
     
       40. The method of  claim 39  wherein said single-stranded nucleic acid ligand is ribonucleic acid. 
     
     
       41. The method of  claim 39  wherein said single-stranded nucleic acid ligand is deoxyribonucleic acid. 
     
     
       42. The method of  claim 37  wherein said protein is not known to bind nucleic acids as part of its biological function. 
     
     
       43. The method of  claim 37  wherein said target molecule is a controlled substance. 
     
     
       44. The method of  claim 37  wherein said target molecule is a metabolite. 
     
     
       45. The method of  claim 37  wherein said sample is a biological substance. 
     
     
       46. The method of  claim 37  wherein said measurement is qualitative. 
     
     
       47. The method of  claim 37  wherein said measurement is quantitative. 
     
     
       48. The method of  claim 37  wherein said candidate mixture is prepared by synthesis from a template comprising a region of conserved sequence and a region of randomized and/or biased sequence. 
     
     
       49. The method of  claim 37  wherein said candidate mixture comprises photoaptamers each comprising a region of conserved sequence and a region of randomized sequence.